Downregulation of LGR5 Expression Inhibits Cardiomyocyte Differentiation and Potentiates Endothelial Differentiation from Human Pluripotent Stem Cells

DOI: 10.1016/j.stemcr.2017.07.006

Authors: Rajneesh Jha; Monalisa Singh; Qingling Wu; Cinsley Gentillon; Marcela K. Preininger; Chunhui Xu

Step 1: iPSC/ESC maintainance

day 0 ~ 1

culture “adherent” human PSC in “MEF-CM” for “1” days. “PSC” should be “downregulated” for “LGR5”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Step 2: Primitive streak 1

day 1 ~ 2

culture “adherent” human PSC in “RPMI 1640” for “1” days. “PSC” should be “downregulated” for “LGR5”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “1” days.

Ideally, “dummy” should express “LGR5”, “TBXT”, “MIXL1” at the end of culture. Ideally, “dummy” should express “TBXT”, “MIXL1” at the end of culture.

Step 3: Primitive streak 2

day 2 ~ 6

culture “adherent” human PSC in “RPMI 1640” for “4” days. “PSC” should be “downregulated” for “LGR5”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “4” days.

Step 4: mesoderm 1

day 6 ~ 8

culture “adherent” human PSC in “RPMI 1640” for “2” days. “PSC” should be “downregulated” for “LGR5”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “dummy” should express “MESP1”, “NKX2-5” at the end of culture. Ideally, “dummy” should express “EOMES”, “GSC”, “TBX6”, “CDX1”, “CDX4”, “MESP1”, “MESP2”, “SOX17”, “HNF3B” at the end of culture.

Step 5: mesoderm 2

day 8 ~ 9 (duration missing in database; assumed 1 day)

culture “unspecified method” unknown species cell in “unspecified medium”.

Ideally, “dummy” should express “HAND1”, “MEF2C”, “NKX2-5”, “TBX5” at the end of culture.

Step 6: endothelial 1

day 9 ~ 11

culture “adherent” human PSC in “M199” for “2” days. “PSC” should be “downregulated” for “LGR5”. For ECM, use “Matrigel” at “N/A” “N/A”. Medium should contain factors shown in the following table, and should be changed once in “2” days.

Ideally, “EC” should be obtained at the end of culture. Ideally, “EC” should express “HHEX”, “TAL1”, “SOX7”, “LMO2”, “PECAM1”, “CD34”, “CDH5” at the end of culture.